Oncolytic viruses represent an emerging group of novel cancer therapeutics that targets cancer cells with defined genetic defects (p53, Rb). Oncolytic viruses eliminate cancer cells through manifestations of cytopathic effects and the viral replication process. Since the same virus does not survive in normal cells, minimal toxicity is produced. Phase I and II clinical trials by us and others showed that oncolytic adenovirotherapy is well tolerated, but has limited efficacy for systemic targeted therapy. This proposal focuses on the late generation, oncolytic adenovirus ONYX-411 with an integrated small interfering RNA payload against the K-ras oncogene (siRNAras). The ras oncogene plays diverse roles in the oncogenetic initiation and maintenance events. K-ras knockdown by the siRNAras transgene transcript is expected to promote tumor cell growth inhibition and apoptosis at the late phase of viral replication. The strategy of restricting siRNA expression to ONYX-411 permissive environment will limit siRNAras knockdown activity to Rb mutant, E2F over-expressing cancer cells, hence obviating the concern of "off target"-siRNA effects in normal cells. We propose to evaluate in 2 phases the hypothesis that ONYX-411 co-expressing siRNAras has enhanced antitumor efficacy without increased toxicity. Our preliminary findings showed that K-ras knockdown by siRNA oligonucleotides led to tumor growth inhibition that was additive to ONYX-411-mediated oncolysis in the human lung cancer line H441. Neither treatment was cytotoxic to normal lung fibroblast growth. Since initial submission, we have successfully produced the ONYX-411-siRNAras construct that was effective for specific mutant K-ras knockdown, promoted tumor cell kill and enhanced viral yield. Milestones for this Phase I proposal are a) Validation of selective viral oncolytic activity; b) Documentation of improved cytopathic outcome and increased viral dissemination in vitro; c) Establishment of toxicity profile of ONYX-411-siRNAras in mice; and d) Validation of an in vivo-applicable safeguard for effective shutdown viral infection and siRNA-related cytopathic effects. Accordingly, Specific Aims for this proposal are: 1. To validate the selective replication, improved cytopathic outcome, and increased viral dissemination of OYX-411-siRNAras in vitro; and 2. To define the in vivo toxicity of ONYX-411- siRNAras. Together with tumor xenograft evaluations and mechanistic analysis described in the upcoming Phase II proposal, these findings will establish the potential systemic applicability of the novel ONYX-411-siRNA "armed" therapeutic virus as a single agent that modulates cancer cell growth within acceptable safety criteria, through the viral cytopathic effects of ONYX-411 and post-transcriptional silencing of the ras oncogene. [unreadable] [unreadable]